The present invention relates generally to bacterial antigens and genes encoding the same. More particularly, the present invention pertains to the construction of a chimeric plasmin binding protein gene comprising the entire S. dysgalactiae gapC coding sequence as well as coding sequences for unique regions from several Streptococcus bacteria species, and the use of the same in vaccine compositions.
Mastitis, an infection of the mammary gland usually caused by bacteria or fungus, results in major economic losses to the dairy industry yearly. Among the bacterial species most commonly associated with mastitis are various species of the genus Streptococcus, including S. aureus, S. uberis, (untypeable), S. agalactiae (Lancefield group B), S. dysgalactiae (Lancefield group C), S. zooepidemicus, and the Lancefield groups D, G., L and N streptococci. Some of those species are contagions (e.g. S. agalactiae), while others are considered environmental pathogens (e.g. S. dysgalactiae and S. uberis). The environmental pathogen S. uberis is responsible for about 20% of all clinical cases of mastitis (Bramley, A. J. and Dodd, F. H. (1984) J. Dairy Res. 51:481-512; Bramley, A. J. (1987) Animal Health Nutrition 42:12-16; Watts, J. L. (1988) J. Dairy Sci. 71:1616-1624); it is the predominant organism isolated from mammary glands during the non-lactating period (Bramley, A. J. (1984) Br. Vet. J. 140:328-335; Bramley and Dodd (1984) J. Dairy Res. 51:481-512; Oliver, S. P. (1988) Am. J. Vet. Res. 49:1789-1793).
Mastitis resulting from infection with S. uberis is commonly subclinical, characterized by apparently normal milk with an increase in somatic cell counts due to the influx of leukocytes. The chemical composition of milk is changed due to suppression of secretion with the transfer of sodium chloride and bicarbonate from blood to milk, causing a shift of pH to a more alkaline level. S. uberis mastitis may also take the form of an acute clinical condition, with obvious signs of disease such as clots or discoloration of the milk and swelling or hardness of the mammary gland. Some cases of the clinical disease can be severe and pyrexia may be present. For a review of the clinical manifestations of S. uberis mastitis, see, Bramley (1991) Mastitis: physiology or pathology. p. 3-9. In C. Burvenich, G. Vandeputte-van Messom, and A. W. Hill (ed.), New insights into the pathogenesis of mastitis. Rijksuniversiteit Gent, Belgium; and Schalm et al. (1971) The mastitis complex-A brief summary. p. 1-3. In Bovine Mastitis. Lea and Febiger, Philadelphia
Conventional antibacterial control methods such as teat dipping and antibiotic therapy are effective in the control of many types of contagious mastitis, but the environmental organisms typically found in all dairy barns are often resistant to such measures. Vaccination is therefore an attractive strategy to prevent infections of the mammary glands, and has been shown to be beneficial in the case of some contagious mastitis pathogens.
The literature is limited regarding vaccination studies with S. dysgalactiae and S. uberis, and variable results have been observed. In some cases, immunization has resulted in increased sensitivity to the specific organism and in other cases strain-specific protection has been obtained.
For example, previous studies have shown that primary infection with S. uberis can considerably reduce the rate of infection following a second challenge with the same strain (Hill, A. W. (1988) Res. Vet. Sci. 44:386-387). Local vaccination with killed S. uberis protects the bovine mammary gland against intramammary challenge with the homologous strain (Finch et al. (1994) Infect. Immun. 62:3599-3603). Similarly, subcutaneous vaccination with live S. uberis has been shown to cause a dramatic modification of the pathogenesis of mastitis with the same strain (Hill et al. (1994) FEMS Immunol. Med. Microbiol. 8:109-118). Animals vaccinated in this way shed fewer bacteria in their milk and many quarters remain free of infection.
Nonetheless, vaccination with live or attenuated bacteria can pose risks to the recipient. Further, it is clear that conventional killed vaccines are in general largely ineffective against S. uberis and S. agalactiae, either due to lack of protective antigens on in vitro-grown cells or masking of these antigens by molecular mimicry.
The current lack of existing mastitis vaccines against S. agalactiae or the contagious streptococcus strains is due at least in part to a lack of knowledge regarding the virulence determinants and protective antigens produced by those organisms which are involved in invasion and protection of the mammary gland (Collins et al. (1988) J. Dairy Res. 55:25-32; Leigh et al. (1990) Res. Vet. Sci. 49: 85-87; Marshall et al. (1986) J. Dairy Res. 53: 507-514).
S. dysgalactiae is known to bind several extracellular and plasma-derived proteins such as fibronectin, fibrinogen, collagen, alpha-II-macroglobulin, IgG, albumin and other compounds. The organism also produces hyaluronidase and fibrinolysin and is capable of adhering to and invading bovine mammary epithelial cells. However, the exact roles of the bacterial components responsible for these phenotypes in pathogenesis is not known.
Similarly, the pathogenesis of S. uberis infection is poorly understood. Furthermore, the influence of S. uberis virulence factors on host defense mechanisms and mammary gland physiology is not well defined. Known virulence factors associated with S. uberis include a hyaluronic acid capsule (Hill, A. W. (1988) Res. Vet. Sci. 45:400-404), hyaluronidase (Schaufuss et al. (1989) Zentralbl. Bakteriol. Ser. A 2711:46-53), R-like protein (Groschup, M. H. and Timoney, J. F. (1993) Res. Vet. Sci. 54:124-126), and a cohemolysin, the CAMP factor, also known as UBERIS factor (Skalka, B. and Smola, J. (1981) Zentralbl Bakteriol. Ser. A 249:190-194), R-like protein, plasminogen activator and CAMP factor. However, very little is known of their roles in pathogenicity.
The use of virulence determinants from Streptococcus as immunogenic agents has been proposed. For example, the CAMP factor of S. uberis has been shown to protect vertebrate subjects from infection by that organism (Jiang, U.S. Pat. No. 5,863,543).
The xcex3 antigen of the group B Streptococci strain A909 (ATCC No. 27591) is a component of the c protein marker complex, which additionally comprises an xcex1 and xcex2 subunit (Boyle, U.S. Pat. No. 5,721,339). Subsets of serotype Ia, II, and virtually all serotype Ib cells of group B streptococci, have been reported to express components of the c protein. Use of the xcex3 subunit as an immunogenic agent against infections by Lancefield Group B Streptococcus infection has been proposed. However, its use to prevent or treat bacterial infections in animals, including mastitis in cattle, has not been studied.
A GapC plasmin binding protein from a strain of Group A Streptococcus has previously been identified and characterized, and its use in thrombolytic therapies has been described (Boyle, et al., U.S. Pat. No. 5,237,050; Boyle, et al., U.S. Pat. No. 5,328,996). However, the use of GapC as an immungenic agent to treat or prevent mastitis was neither described nor suggested.
The group A streptococcal M protein is considered to be one of the major virulence factors of this organism by virtue of its ability to impede attack by human phagocytes (Lancefield, R. C. (1962) J. Immunol 89:307-313). The bacteria persist in the infected tissue until antibodies are produced against the M molecule. Type-specific antibodies to the M protein are able to reverse the antiphagocytic effect of the molecule and allow efficient clearance of the invading organism.
M proteins are one of the key virulence factors of Streptococcus pyogenes, due to their involvement in mediating resistance to phagocytosis (Kehoe, M. A. (1991) Vaccine 9:797-806) and their ability to induce potentially harmful host immune responses via their superantigenicity and their capacity to induce host-cross-reactive antibody responses (Bisno, A. L. (1991) New Engl. J. Med. 325:783-793; Froude et al. (1989) Curr. Top. Microbiol Immunol. 145:5-26; Stollerman, G. H. (1991) Clin. Immunol. Immunopathol. 61:131-142).
However, obstacles exist to using intact M proteins as vaccines. The protein""s opsonic epitopes are extremely type-specific, resulting in narrow, type-specific protection. Further, some M proteins appear to contain epitopes that cross react with tissues of the immunized subject, causing a harmful autoimmune response (See e.g., Dale, J. L. and Beached, G. H. (1982) J. Exp. Med 156:1165-1176; Dale, J. L. and Beached, G. H. (1985) J. Exp. Med. 161:113-122; Baird, R. W., Bronze, M. S., Drabs, W., Hill, H. R., Veasey, L. G. and Dale, J. L. (1991) J. Immun. 146:3132-3137; Bronze, M. S. and Dale, J. L. (1993) J. Immun 151:2820-2828; Cunningham, M. W. and Russell, S. M. (1983) Infect. Immun. 42:531-538).
An octavalent M protein vaccine has been constructed and was tested for protective immunogenicity against multiple serotypes of group A streptococci infection in rabbits. However, the immune response obtained was serotype-specific, conferring protection only against those bacterial strains exhibiting the M protein epitopes present in the chimeric protein (Dale, J. B., Simmons, M., Chiang, E. C., and Chiang, E. Y. (1996) Vaccine 14:944-948).
Chimeric proteins containing three different fibronectin binding domains (FNBDs) derived from fibronectin binding proteins of S. dysgalactiae and Staphylococcus aureus have been expressed on the surface of Staph. carnosus cells. In the case of one of these proteins, intranasal immunizations with live recombinant Staph. carnosus cells expressing the chimeric protein on their surface resulted in an improved antibody response to a model immunogen present within the chimeric surface protein.
A chimeric Protein G molecule (a type III Fc binding protein specific for the Fc region of all subclasses of IgG antibody molecules) is known, but its use as an immunogenic agent has not been described or suggested (Bjorck, et al. (1992) U.S. Pat. No. 5,108,894).
Until now, the protective capability of GapC multiple epitope fusion proteins has not been studied.
Accordingly, the present invention provides GapC multiple epitope fusion proteins and polynucleotides encoding the same. In one embodiment, the invention is directed to a multiple epitope fusion polypeptide comprising the general structural formula (I):
xe2x80x83(A)xxe2x80x94(B)yxe2x80x94(C)zxe2x80x83xe2x80x83(I)
wherein
(I) is a linear amino acid sequence;
B comprises an amino acid sequence containing at least five amino acids which amino acids correspond to an antigenic determinant of a GapC protein;
A and C each comprise an amino acid sequence that is
(i) different from B,
(ii) different from the other, and
(iii) an amino acid sequence containing at least five amino acids, which
amino acid sequence corresponds to an antigenic determinant of a GapC protein wherein said antigenic determinant is not adjacent to B in nature;
y is an integer of 1 or more; and
x and z are each independently integers wherein x+z is 1 or more.
In certain embodiments, the multiple epitope fusion polypeptide further comprises a signal sequence and/or a transmembrane sequence. Further, A, B, and/or C of the multiple epitope fusion polypeptide may linked by one or more spacer sequences, wherein the spacers
(i) are amino acid sequences of from 1 to 1,000 amino acids, inclusive;
(ii) can be the same or different as A, B, or C; and
(iii) can be the same or different as each other.
In certain embodiments, A, B, and C each comprise epitopes from one or more species of bacteria, such as from one or more bacterial species of the genus Streptococcus, including but not limited to one or more bacterial species selected from the group consisting of Streptococcus dysgalactiae, Streptococcus agalactiae, Streptococcus uberis, Streptococcus parauberis, and Streptococcus iniae. 
In yet another embodiment, A, B, and C each comprise amino acid sequences selected from the group consisting of
(a) the amino acid sequence shown at about amino acid positions 61 to 81, inclusive, of FIGS. 1 through 5, or any amino acid sequence having at least about 80% identity thereto;
(b) the amino acid sequences shown at about amino acid positions 102 to 112, inclusive, of FIGS. 1 through 5, or any amino acid sequence having at least about 80% identity thereto;
(c) the amino acid sequences shown at about amino acid positions 165 to 172, inclusive, of FIGS. 1 through 5, or any amino acid sequence having at least about 80% identity thereto;
(d) the amino acid sequences shown at about amino acid positions 248 to 271, inclusive, of figures through 5, or any amino acid sequence having at least about 80% identity thereto; and
(e) the amino acid sequences shown at about amino acid positions 286 to 305, inclusive, of FIGS. 1 through 5, or any amino acid sequence having at least about 80% identity thereto.
In another embodiment, the multiple epitope fusion polypeptide comprises the amino acid sequence depicted in FIG. 6 (SEQ ID NO: 22).
In yet further embodiments, the invention is directed to polynucleotide sequences encoding the multiple epitope fusion polypeptide sequence described above or compliments thereof, as well as recombinant vectors comprising the polynucleotide, host cells comprising the recombinant vectors and methods of recombinantly producing the polypeptides.
In another embodiment, the invention is directed to a vaccine composition comprising a pharmaceutically acceptable vehicle and a multiple epitope fusion polypeptide as described above. In certain embodiments, the vaccine compositions comprise an adjuvant.
In still a further embodiment, the invention is directed to a method of producing a vaccine composition comprising the steps of
(1) providing the multiple epitope fusion polypeptide; and
(2) combining the polypeptide with a pharmaceutically acceptable vehicle.
In another embodiment, the invention is directed to a method of treating or preventing a bacterial infection in a vertebrate subject comprising administering to the subject a therapeutically effective amount of a vaccine composition as described above.
In certain embodiments, the bacterial infection is a streptococcal infection. Further, the bacterial infection may cause mastitis.
In yet another embodiment, the invention is directed to a method of treating or preventing a bacterial infection in a vertebrate subject comprising administering to the subject a therapeutically effective amount of a polynucleotide as described herein.
In certain embodiments, the bacterial infection is a streptococcal infection. Further, the bacterial infection may cause mastitis.
In further embodiments, the invention is directed to antibodies directed against the above multiple epitope fusion polypeptides. The antibodies may be polyclonal or monoclonal.
In another embodiment, the invention is directed to a method of detecting Streptococcus antibodies in a biological sample, comprising:
(a) reacting said biological sample with a multiple epitope fusion polypeptide under conditions which allow said Streptococcus antibodies, when present in the biological sample, to bind to said sequence to form an antibody/antigen complex; and
(b) detecting the presence or absence of said complex, and thereby detecting the presence or absence of Streptococcus antibodies in said sample.
In still a further embodiment, the invention is directed to an immunodiagnostic test kit for detecting Streptococcus infection. The test kit comprises a multiple epitope fusion polypeptide as described herein and instructions for conducting the immunodiagnostic test.
These and other embodiments of the subject invention will readily occur to those of skill in the art in view of the disclosure herein.